Research ArticleOxidative Stress in Animal Models of Acute and ChronicRenal Failure

Marianna Gyurászová,1 Alexandra Gaál Kovalčíková,1,2 Emese Renczés ,1

Katarína Kmeťová,1 Peter Celec ,1,3,4 Janka Bábíčková ,1,5 and Ľubomíra Tóthová 1

1Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia2Department of Pediatrics, National Institute of Children’s Diseases and Faculty of Medicine, Comenius University,Bratislava, Slovakia3Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia4Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia5Department of Clinical Medicine, University of Bergen, Bergen, Norway

Correspondence should be addressed to Janka Bábíčková; jana.babickova@gmail.com

Received 25 November 2018; Revised 27 December 2018; Accepted 19 January 2019; Published 11 February 2019

Guest Editor: Christos Chadjichristos

Copyright © 2019 Marianna Gyurászová et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction. Kidney disease is a worldwide health and economic burden, with rising prevalence. The search for biomarkers forearlier and more effective disease screening and monitoring is needed. Oxidative stress has been linked to both, acute kidneyinjury (AKI) and chronic kidney disease (CKD). The aim of our study was to investigate whether the concentrations of systemicmarkers of oxidative stress and antioxidant status are affected by AKI and CKD, and to identify potential biomarkers. Methods.In adult male Wistar rats, AKI was induced by bilateral nephrectomy, and CKD was induced by 5/6 nephrectomy. Blood wascollected 48 hours after surgery in AKI and 6 months after surgery in CKD. Advanced oxidation protein products (AOPP),thiobarbituric acid reactive substances (TBARS), advanced glycation end products (AGEs), fructosamine, total antioxidantcapacity (TAC), and ferric reducing antioxidant power (FRAP) were measured. Results. Impaired renal function was confirmedby high concentrations of plasma creatinine and urea in AKI and CKD animals. AOPP and fructosamine were higher by 100%and 54% in AKI, respectively, and by 100% and 199% in CKD, respectively, when compared to corresponding control groups.Similarly, there was approximately a twofold increase in AGEs (by 92%) and TAC (by 102%) during AKI. In CKD,concentrations of FRAP, as an antioxidative status marker, were doubled (by 107%) when compared to the control group, butconcentration of TAC, another marker of antioxidative status, did not differ between the groups. Conclusions. AKI and CKD ledto increased systemic oxidative stress. AOPP and fructosamine could be considered potential biomarkers for both, acute andchronic kidney damage. On the other hand, AGEs, TAC, and FRAP seem to be disease specific, which could help to differentiatebetween acute and chronic kidney injuries. However, this needs further validation in clinical studies.

1. Introduction

Kidney disease can be classified into two types: acute kidneyinjury (AKI) and chronic kidney disease (CKD). The globalprevalence of both forms of kidney disease is rising continu-ously, in part due to the aging population and in part due to aglobal increase in the prevalence of hypertension and diabe-tes. AKI is characterized by a sudden loss of kidney functionwithin 7 days. CKD develops due to a structural or functionalkidney irregularity that persists for at least 3 months. Both

AKI and CKD result in the accumulation of toxic end prod-ucts of nitrogen metabolism and creatinine in the blood[1–3]. Research is focused on the search for more effectiveand less costly therapies of AKI and end-stage CKD. Moreimportantly, the search for screening methods of earlier detec-tion and more effective disease monitoring is ongoing [4].

Oxidative stress is a state of imbalance between thegeneration of prooxidants and the number of antioxidantspresent in favor of prooxidants. Free radicals and nonradicaloxidants alter biomolecules, mainly lipids, proteins, and

HindawiDisease MarkersVolume 2019, Article ID 8690805, 10 pageshttps://doi.org/10.1155/2019/8690805

nucleic acids, ultimately leading to cell death. Mitochondriaprovide the main energy source for cells. In the kidney, renaltubular cells are especially rich in mitochondria, because thereabsorption of solutes is highly energy demanding. Renaltubules are particularly vulnerable to oxidative stress anddamage, since mitochondria are one of the main sites ofintracellular free radical production via the respiratorychain and NADPH oxidases [5–7]. Increased production ofprooxidants is involved in many pathological pathways ofAKI and CKD [8].

In CKD, impaired mitochondrial function and enhancedmitochondrial reactive oxygen species (ROS) has been pro-posed as one of the causes of elevated oxidative stress [9].Increased production of mitochondrial ROS was shown inthe kidneys of diabetic mice [10–12]. In AKI, oxidativedamage and decreased antioxidant status can develop in therenal tissue due to ischemia and toxic damage [13–15]. Also,oxidative stress is a key factor in the pathogenesis ofrhabdomyolysis-induced myoglobinuric AKI [16]. Anothersource of oxidative stress in later stages of CKD may be thepresence of uremic toxins and dysregulated metabolic wastedisposal [17–23]. In patients with CKD, uremia-specific riskfactors, including volume expansion, chronic inflammation,anemia, or a microinflammatory state, are associated withsystemic oxidative stress that in turn could cause inflamma-tion and further tissue damage [6]. Thus, oxidative stresscan affect the progression of renal disease as well in a bidirec-tional manner. It can aggravate inflammation, contribute tothe development of fibrosis, via enhanced inflammationand trigger signaling pathways leading to renal tubular celldeath. Fibrosis and inflammation might increase further for-mation of ROS [24]. According to the above-mentionedstudies, it has been shown that oxidative stress is stronglyassociated with AKI and CKD and their complications. How-ever, it is still questionable whether an elevated production ofprooxidants is the only reason of oxidative stress. Uremictoxins bolster inflammation along with oxidative stress bypriming polymorphonuclear leukocytes and triggering aninnate immune response. Uric acid synthesis can furtherpromote oxidative stress through the activity of xanthineoxidoreductase. On the other hand, the uric acid itselfcan act as an antioxidant [25–27].

Thus, oxidative stress and antioxidant status markersneed to be analyzed in detail. Oxidative status is commonlyassessed in the plasma by measuring the concentrations ofdamaged biomolecules by free radicals and other oxidants.Lipid peroxidation is routinely measured by the thiobarbitu-ric acid-reacting substance (TBARS) method [28]. For theevaluation of protein oxidation, advanced oxidation proteinproducts (AOPP) are assessed [29]. Advanced glycation endproducts (AGEs) along with fructosamine are formed aslate-stage products of nonenzymatic glycation of aminogroups of proteins by the carbonyl compound of sugars,and they are used as markers of carbonyl stress [30]. Totalantioxidant capacity (TAC) and ferric reducing antioxidantpower (FRAP) are commonly used methods for the assess-ment of the antioxidant status [31, 32].

To our knowledge, only very few studies describing oxi-dative stress and antioxidant status markers in detail in both

AKI and CKD have been published. The most recent onesuggests that salivary AOPP could be a suitable marker forthe detection of CKD in children with 92% sensitivity andspecificity [33]. Since AOPP are nonspecific, a panel of oxida-tive stress markers would be more helpful in the detection ofCKD or AKI. Therefore, the main goal of this study was toexperimentally determine the effect of acute kidney injuryand chronic kidney disease on the systemic oxidative status.In addition, AKI and CKDwere analyzed in parallel to searchfor possible differences between the two conditions, whichmight prove useful in the differentiation between progressiveCKD and AKI as a complication of CKD or alone.

2. Methods

2.1. Animals. Twelve-week-old adult male Wistar rats wereused in this experiment (n = 40 in total, weighing 294 ± 79grams, Anlab, Prague, Czech Republic). Rats were housedin standard cages with wood chip bedding, in a room withan ambient temperature of 22 ± 1°C, 40-50% humidity, anda 12/12-hour light/dark cycle. All rats had ad libitum accessto standard rodent chow and tap water throughout theexperiment. This study was approved by the Ethics Com-mittee of the Institute of Pathophysiology, ComeniusUniversity, and was carried out according to relevantnational legislation.

2.2. Modeling of Acute Kidney Injury. Bilateral nephrectomywas performed to model AKI. Rats were divided into twogroups, a bilateral nephrectomy group (BNex, n = 11) and asham group (BNex sham, n = 6). The animals were bilaterallynephrectomised in one surgical session, as describedpreviously [34]. Animals were anesthetized by ketamine(100mg/kg, Richter Pharma AG, Wels, Austria) and xylazine(10mg/kg, Ecuphar N.V., Oostkamp, Belgium) administeredby intraperitoneal injection. A midline incision was made inthe BNex group; the kidneys were exposed and decapsulated.Renal pedicles were tied offwith a suture, and the kidneys wereremoved. The incision was closed with an absorbable suture.Animals in the sham group underwent sham surgery. Bothkidneys of the rats in this group were decapsulated. Animalswere sacrificed 48 hours after surgery, under general anesthe-sia. Blood was collected into EDTA-coated blood collectiontubes (Sarstedt, Numbrecht, Germany) from the abdominalaorta. Plasma was obtained by centrifugation (5000 g for 5minutes) and stored at -20°C until further analysis.

2.3. Modeling of Chronic Kidney Disease. To model CKD, 5/6nephrectomy was conducted. Rats were divided into twogroups, a 5/6 nephrectomised (5/6 Nex, n = 14) and a shamgroup (5/6 Nex sham, n = 9). The animals in the 5/6 Nexgroup underwent subtotal (5/6) nephrectomy in two surgicalsteps, as reported previously [35]. Briefly, animals were anes-thetized by ketamine (100mg/kg, Richter Pharma AG, Wels,Austria) and xylazine (10mg/kg, Ecuphar N.V., Oostkamp,Belgium) administered by intraperitoneal injection. A mid-line incision was made on the left side, and the left kidneywas exposed. The kidney was decapsulated, and the upperand lower kidney poles were removed. Bleeding was stopped

2 Disease Markers

by Gelaspon (Chauvin Ankerpharm GmbH, Rudolstadt,Germany), and the incision was closed with an absorbablesuture. After the recovery period (14 days later), a similarincision was made on the right side, the renal vessels wereligated, and the decapsulated kidney was removed. Animalsin the 5/6 Nex sham group underwent sham surgery. Thekidneys of the rats in this group were decapsulated at the timeof the first surgery. Animals were sacrificed 6 months aftersurgery under general anesthesia. Animals were placed inmetabolic cages (4 hours) for urine collection. Plasma sam-ples were obtained in the same manner as described in theAKI model.

2.4. Biochemical Analysis. Three hundred and fifty microli-ters of plasma were used to measure plasma creatinine, urea,and albuminuria concentrations using the Biolis 24i Pre-mium automated clinical analyzer (Tokyo Boeki MedicalSystem Ltd., Tokyo, Japan) [36]. The principle of urea mea-surement is based on the hydrolysis of urea by urease to formammonium and carbonate. The second step is based on thereaction 2-oxoglutarate that reacts with ammonium in thepresence of glutamate dehydrogenase (GLDH) and the coen-zyme NADH. This reaction produces L-glutamate [37]. Themeasurement of creatinine is based on its reaction with picricacid in alkaline conditions that results in a reddish complex[38]. The plasma concentrations of kidney injury molecule1 (KIM-1) were measured using the commercial rat KIM-1ELISA kit (R&D Systems Inc., Abingdon, UK) according tothe manufacturer’s protocol.

2.5. Oxidative Stress and Antioxidant Status Analysis.Markers of oxidative stress and antioxidant status weremeasured in plasma samples using a Synergy H1 multimodemicroplate reader (BioTek Instruments, Inc., Winooski,VT, USA).

TBARS were measured by pipetting 20μl of samples orstandards (1,1,3,3-tetraethoxypropane), 30 μl of distilledwater, 20μl of 0.67% thiobarbituric acid, and 20μl of glacialacetic acid in a microtiter plate. The plates were mixed andincubated for 45 minutes at 95°C. 100μl of n-butanol wasadded into the samples, and the plates were centrifuged(2000 g/10min/4°C). Seventy microliters of the upper phasewas transferred into a new microtiter plate, and fluorescencewas measured at ex = 515 nm and λem = 535 nm. AOPP wasassessed by mixing 200 μl of samples and standards (chlo-ramine T mixed with potassium iodide) with 20μl of gla-cial acetic acid for 2 minutes. Absorbance was measured at340nm. AGEs were measured by pipetting 20μl of sampleor standards (AGE-BSA) and 180μl of PBS into a darkmicrotiter plate, vortexing and measuring fluorescence at

ex = 370 nm and λem = 440 nm. For fructosamine measure-ment, 20 μl of the samples and standards (16mmol/l1-deoxy-morpholino-D-fructose) was mixed with 100 μlof 0.25mmol/l nitroblue tetrazolium containing 1mmol/lnitroblue tetrazolium and 0.1mol/l sodium carbonatebuffer (pH = 10 35). Samples were incubated at 37°C for15 minutes. Absorbance was measured at 530nm.

For TAC measurement, samples were mixed with acetatebuffer (pH = 5 8). Absorbance was measured at 660nm as

blank. When the ABTS solution (2.2′-azino-bis(3-ethyl-benzthiazoline-6-sulphonic acid with acetate buffer)) wasadded, the absorbance was measured again at 660nm. Theblank absorbance values were subtracted from the valuesobtained by the second measurement. For FRAP assessment,200 μl of warmed (37°C) FRAP reagent (containing acetatebuffer (pH = 3 6), tripyridyl-s-triazine, FeCl3∗6H2O, andwater) was pipetted into a microtiter plate, and absorbancewas measured as blank. Afterwards, 20 μl of samples andstandards (100mmol/l FeSO4∗7H2O) was added. Absor-bance was measured again at 530nm. The blank absorbancevalues were subtracted from the values obtained by the sec-ond measurement [39].

For the measurement of proteins, 10μl of the samplesand standards (bovine serum albumin) was mixed with200μl of the working solution (bicinchoninic acid andcopper sulphate, 49 : 1 ratio, respectively). The plate wasincubated at 37°C for 30 minutes. After cooling, absor-bance was measured at 562 nm. All markers measured inplasma were normalized to plasma protein concentrations.Markers measured in urine were normalized to urinarycreatinine concentrations.

2.6. Statistical Analysis. GraphPad Prism 5.0 (GraphPadSoftware, San Diego, CA, USA) was used for the statis-tical analyses. After testing for normality with theD’Agostino-Pearson omnibus test, data were analyzedwith the Mann–Whitney U test. The Spearman correla-tion test and linear regression were used to evaluatethe linear associations between quantitative variables. Avalue of p < 0 05 was considered statistically significant.

3. Results

Concentrations of creatinine in the plasma of the BNexgroup were 20-fold higher when compared to the BNex shamgroup (Figure 1(a); U = 0, p < 0 001). Blood urea in the BNexgroup was significantly higher when compared to the BNexsham group by 484% (Figure 1(b); U = 0, p < 0 001). Plasmacreatinine concentrations in the 5/6 Nex group were signifi-cantly higher than in the 5/6 Nex sham group by 48%(Figure 1(c); U = 18, p < 0 01). Blood urea was 2-fold higherin the 5/6 Nex group than in the 5/6 Nex sham group(Figure 1(d); U = 10, p < 0 001). One animal in the BNexgroup did not show elevated levels of plasma creatinine(37.56μmol/l), nor urea (11.70mmol/l), which would com-ply with the modeling of AKI and was thus removed fromfurther analyses.

There were no differences between the BNex and theBNex sham groups in TBARS concentrations (Figure 2(a)).In plasma TBARS, no significant differences were foundbetween the 5/6 Nex and the 5/6 Nex sham groups either(Figure 3(a)). Plasma AOPP concentrations were 2-foldhigher in the BNex group, when compared to the BNex shamgroup (Figure 2(b); U = 9, p < 0 05). In plasma AOPP, asignificant difference was found between the 5/6 Nex andthe 5/6 Nex sham groups, the first being higher by 102%(Figure 3(b); U = 28, p < 0 05).

3Disease Markers

AGE concentrations were 2-fold higher in the plasma ofthe BNex group, when compared to the BNex sham group(Figure 2(c); U = 0, p < 0 01). No significant differences werefound in plasma AGE concentration between the 5/6 Nexand the 5/6 Nex sham groups (Figure 3(c)). Fructosamineconcentrations differed significantly between the BNex andthe BNex sham group as well, the BNex group being higherby 54% (Figure 2(d); U = 4, p < 0 01). In plasma fructosa-mine, a significant difference was found between the 5/6Nex and the 5/6 Nex sham groups. The 5/6 Nex group had3-fold higher fructosamine concentrations (Figure 3(d);U = 10, p < 0 01). Between the BNex and the BNex shamgroups, there was a significant difference in TAC, theBNex being 2-fold higher (Figure 2(e); U = 5, p < 0 05).TAC concentrations in plasma did not differ betweenthe 5/6 Nex and the 5/6 Nex sham groups (Figure 3(e)).There were no differences between the BNex and the BNexsham groups in FRAP concentrations (Figure 2(f)). Inplasma FRAP, a significant difference was found betweenthe 5/6 Nex and the 5/6 Nex sham groups, the first being2-fold higher (Figure 3(f); U = 22, p < 0 05).

Additionally, to the plasma concentrations, TBARS,AOPP, fructosamine, and FRAP were all measurable in theurine of 5/6 Nex rats. Of these, AOPP differed most signifi-cantly from the sham group. AOPP in 5/6 Nex were 2-foldhigher in urine when compared to 5/6 Nex sham group

(Figure 4(b); U = 7, p < 0 001). TBARS, fructosamine, andFRAP in urine were increased in the 5/6 Nex group byapproximately 50% (Figures 4(a), 4(c), and 4(d); U = 40, 34,and 32, respectively, p < 0 05 for all three markers) whencompared to the control group.

In 5/6 Nex as a model of CKD, urinary AOPP signifi-cantly and positively correlated with urinary KIM-1(r = 0 70; p < 0 05). Also, urinary fructosamine and FRAPsignificantly correlated with ACR (r = 0 72 and r = 0 82;p < 0 05, respectively) and albuminuria (r = 0 62 and r =0 82; p < 0 05, respectively), but neither with plasma norwith urinary KIM-1 (Table 1).

4. Discussion

The results of this experiment confirmed the associationbetween elevated systemic oxidative stress and acute andchronic renal failure. Although proteins are relatively resis-tant to damage by prooxidants, AOPP concentrations were2-fold higher in rats with both acute and chronic renal failurecompared to their sham-operated counterparts. A 2-foldincrease in AOPP was present not only in plasma but alsoin the urine of CKD rats. These findings are in line withprevious studies in AKI and CKD patients [40, 41]. Ithas been proposed that AOPP not only are associated withCKD but also have a pathogenic role in CKD progression

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Figure 1: Renal function parameters in rats with acute and chronic kidney injury and sham-operated rats. (a) Plasma creatinine in bilaterallynephrectomised (BNex) and sham (BNex sham) rats, (b) plasma urea in BNex and BNex sham rats, (c) plasma creatinine in 5/6nephrectomised (5/6 Nex) and sham (5/6 Nex sham) rats, and (d) plasma urea in 5/6 Nex and 5/6 Nex sham rats. ∗∗p < 0 01, ∗∗∗p < 0 001.

4 Disease Markers

through cellular and molecular mechanisms. These mech-anisms include triggering a cascade of signaling events thatlead to superoxide generation, NF-κB activation, the over-production of extracellular matrix, apoptosis of podocytes,endothelial inflammation, and monocyte activation [42, 43].Moreover, elevated AOPP were found to be associated withpoor prognosis of patients with IgA nephropathy [44]. Onthe other hand, during CKD, the excessive glomerularprotein leakage might contribute to the absolute numbersof AOPP.

This study found no differences between 5/6 Nex orBNex animals and the sham groups in oxidized lipid

concentrations measured by TBARS in plasma, contrary toother studies. A previous study showed that products of lipidperoxidation could be increasing in AKI in a time-dependentmanner. It could be argued that these damaged lipids wouldhave increased should the experiment lasted longer,although, in the mentioned studies, lipid peroxidation prod-ucts were already significantly higher after 48 hours in ratswith AKI [45, 46]. On the other hand, urinary TBARS werehigher in CKD rats, suggesting their clearance from circula-tion despite reduced renal function. Unfortunately, due tothe anuria of the BNex rats, we were unable to determinethe urinary levels of TBARS in the acute setting.

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Figure 2: Oxidative stress and antioxidant status markers in the plasma of bilaterally nephrectomised (BNex) and sham (BNex sham) rats. (a)TBARS: thiobarbituric acid reactive substances, (b) AOPP: advanced oxidation protein products, (c) AGEs: advanced glycation end products,(d) fructosamine, (e) TAC: total antioxidant capacity, and (f) FRAP: ferric reducing antioxidant power. ∗p < 0 05, ∗∗p < 0 01.

5Disease Markers

Products of carbonyl stress, such as fructosamine andAGEs, are produced during aging and accumulate in circula-tion and tissues. Kidneys play an important part in their dis-posal, and fructosamine and AGEs have been shown to beelevated when kidney function is compromised [47, 48].The results of this experiment showed that the concentrationof fructosamine was higher in AKI and CKD, but AGEs werehigher only in the AKI model. This could indicate that theremaining kidney function in the mild 5/6 Nex model ofCKD was sufficient to filter AGEs from circulation. The rea-sons of the accumulation of AGEs during uremia are not fullyunderstood. Increased concentration of circulating AGEs in

patients with kidney diseases may result from an increasedproduction due to uremia-related consequences or decreasedrenal disposal [49]. According to previous results, low molec-ular weight AGEs are easily removed from the body by renalclearance [50]. Thus, it seems that a partial maintenance oflow molecular weight AGE disposal by the damaged kidneysprevented the elevation of plasma AGE concentrations in 5/6Nex. However, it has also been shown that one fraction ofplasma AGEs is linked to binding proteins, mainly albuminin uremic patients. Glomerular filtration or dialysis doesnot remove these proteins. Thus, accumulation of highermolecular weight AGEs could not be explained by decreased

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Figure 3: Oxidative stress and antioxidant status markers in the plasma of 5/6 nephrectomised (5/6 Nex) and sham (5/6 Nex sham) rats. (a)TBARS: thiobarbituric acid reactive substances, (b) AOPP: advanced oxidation protein products, (c) AGEs: advanced glycation end products,(d) fructosamine, (e) TAC: total antioxidant capacity, and (f) FRAP: ferric reducing antioxidant power. ∗p < 0 05, ∗∗p < 0 01.

6 Disease Markers

renal clearance [51]. In addition, the elevated urinary levelsof fructosamine in 5/6 Nex rats confirm their clearance viathe kidneys.

The results also showed that the concentrations of anti-oxidant status markers were higher in the 5/6 Nex and BNexgroups than in controls—more precisely, FRAP was higher in

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Figure 4: Oxidative stress and antioxidant status markers in the urine of 5/6 nephrectomised (5/6 Nex) and sham (5/6 Nex sham) rats. (a)TBARS: thiobarbituric acid reactive substances, (b) AOPP: advanced oxidation protein products, (c) fructosamine, and (d) FRAP: ferricreducing antioxidant power. ∗p < 0 05, ∗∗∗p < 0 001.

Table 1: Correlation coefficients (Spearman) between plasma markers, thiobarbituric acid reactive substances (TBARS), advanced oxidationprotein products (AOPP), advanced glycation end products (AGEs), fructosamine, total antioxidant capacity (TAC), and ferric reducingantioxidant power (FRAP), and urinary markers, TBARS, AOPP, fructosamine, FRAP, plasma creatinine, blood urea nitrogen (BUN),urinary kidney injury molecule-1 (uKIM-1), plasma kidney injury molecule-1 (pKIM-1), albumin/creatinine ratio of urine (ACR), andalbuminuria in 5/6 nephrectomised (CKD model) and sham rats.

Plasma creatinine(μmol/l)

BUN(mmol/l)

pKIM-1(pg/ml)

uKIM-1(pg/ml)

ACR(g/mol)

Albuminuria(mg/day)

Plasma markers of OS

TBARS (μmol/g) -0.42 n.s. -0.38 n.s. 0.06 n.s. -0.30 n.s. 0.33 n.s. 0.39 n.s.

AOPP (μmol/g) -0.12 n.s. 0.05 n.s -0.15 n.s. -0.05 n.s. -0.07 n.s. -0.08 n.s.

AGEs (g/g) -0.60∗ -0.38 n.s. -0.38 n.s. -0.62∗ -0.32 n.s -0.11 n.s.

Fructosamine (mmol/g) 0.10 n.s. 0.17 n.s. -0.43 n.s. -0.05 n.s. -0.06 n.s. 0.07 n.s.

TAC (μmol/g) 0.01 n.s. 0.10 n.s. 0.28 n.s. 0.17 n.s. 0.0 n.s. -0.08 n.s.

FRAP (μmol/g) 0.07 n.s. 0.10 n.s. 0.06 n.s. 0.12 n.s. 0.14 n.s. 0.12 n.s.

Urinary markers of OS

TBARS (μmol/mmol) 0.23 n.s. 0.44 n.s. -0.06 n.s. 0.26 n.s. 0.30 n.s. 0.01 n.s.

AOPP (μmol/mmol∗g) 0.23 n.s. 0.06 n.s. 0.38 n.s. 0.70∗ 0.51 n.s. -0.10 n.s.

Fructosamine(mmol/mmol)

-0.31 n.s. -0.32 n.s. 0.13 n.s. 0.17 n.s. 0.72∗ 0.62∗

FRAP (μmol/mmol) -0.33 n.s. -0.28 n.s. 0.49 n.s. 0.32 n.s. 0.82∗∗ 0.69∗

∗p < 0 05, ∗∗p < 0 01.

7Disease Markers

the 5/6 Nex group, and TAC was found to be elevated in theBNex group when compared to sham animals. These resultsare in line with a different study showing higher TAC in a ratmodel of AKI than in sham-operated animals [52]. Anotherstudy involving CKD patients has shown higher FRAPconcentrations in patients than in controls [53]. In addition,urinary FRAP levels were also elevated in 5/6 Nex, confirm-ing the situation observed in plasma. Although we cannotfully explain the difference between AKI and CKD, this couldbe potentially used as a differentiating marker between AKIand CKD. It is assumed that the antioxidant activity rose asan answer to present systemic oxidative stress in both diseasestates. Paradoxically, the antioxidant status may increase inrenal disease because of the accumulation of uremic toxinswith scavenging capacity, such as indole derivatives and hip-purate [48, 52]. In addition, the difference might be the resultof the differences of the measured substances between thetwo methods. While TAC includes the SH/thiol groups pres-ent in proteins and reduced glutathione, these are notdetected by the FRAP method. Due to the complete anuriain the BNex rats compared to the known proteinuria in the5/6 Nex model, the higher TAC in BNex might be attributedto a higher amount of small molecular weight proteins in theplasma of BNex rats [54].

To further address the applicability of the oxidative stressmarkers in renal disease, we correlated them with thestandard plasmatic and urinary markers of renal functionand injury.

In CKD, we found a negative correlation between plasmaAGEs vs. plasma creatinine and uKIM-1. Whether thisobservation is of any prognostic or diagnostic value is cur-rently unknown. In the urine, there was a positive correlationbetween AOPP and uKIM-1, suggesting that urinary AOPPmight closely reflect proximal tubular injury. On the otherhand, urinary fructosamine and FRAP correlated with ACRand albuminuria, but not with uKIM-1, suggesting that theymight more closely reflect glomerular injury. However,further studies are needed to prove these assumptions.

The limitation of this study is that the urinary concen-tration of oxidative stress and antioxidant status markerscould not be assessed in BNex rats, due to anuria whichis typical for this model [55]. Further studies will be aimedat the models with residual kidney function, which are notcompletely anuric.

In conclusion, this study confirms the association of AKIand CKD with elevated systemic oxidative stress markers.Furthermore, to our knowledge, this study is the first todescribe this particular set of oxidative stress and antioxidantstatus markers measured parallel in AKI and CKD. It showsthat oxidative damage to proteins is apparent in both diseasestates in plasma and in urine. Nevertheless, we do not reportelevated lipid peroxidation in kidney disease in plasma, nei-ther in AKI nor in CKD. Interestingly, in the urine, we foundelevated markers of lipid peroxidation in CKD, which mightsuggest urine as a superior fluid for the determination of lipidperoxidation in CKD. Carbonyl stress is apparent by anincrease in fructosamine in both disease states, but the rem-nant kidney function in CKD appears to be sufficient toexcrete AGEs of lower molecular weight. Antioxidant status

can also increase in AKI and CKD, likely due to an increasein uremic toxins with antioxidant capacity. However differ-ent antioxidant systems probably take part in increasedoxidative stress during CKD and AKI. Future studiesshould be focused on AOPP and fructosamine as potentialbiomarkers, and data on their dynamics and urinary con-centrations in AKI should be gathered. Further clarifica-tion of different antioxidant mechanisms during AKI andCKD is needed.

Data Availability

The Excel or GraphPad data used to support the findingsof this study are available from the corresponding authorupon request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This study was funded by the Ministry of Education, Science,Research and Sport of the Slovak Republic (grant numberVEGA 1/0234/18) and by the Momentum (Career Develop-ment Program for Young Researchers) of the University ofBergen, Norway.

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